The overall objective of this revised second competitive renewal is to continue our investigations on the role of photodynamic therapy (PDT) in treating localized infections. PDT employs non-toxic photosensitizers (PS) and harmless visible light (frequently red light for increased tissue penetration) that combine in the presence f oxygen to produce reactive oxygen species that damage biological molecules such as proteins, lipids and nucleic acids and subsequently cause cell death. In the two funding periods of this grant we have synthesized and characterized several novel highly active antimicrobial PS and demonstrated their effectiveness in treating mouse models of infected wounds, burns and abscesses. In some cases PDT can save mice from a certain death due to sepsis that develops from an untreated localized infection. A combination of the appropriate molecular design of the PS, together with topical or local application of the PS to the infected area and a short drug-ligh interval allows high selectivity for microbial cells compared to the surrounding host cells. The broad motivation for this line of research is the relentless worldwide increase in antibiotic resistance amongst pathogenic microbes, and it has been found that multi-antibiotic resistant strains are in general as sensitive to photodynamic inactivation (PDI) as nave strains, and moreover that microbial cells are unable to develop resistance to PDI. Aim 1 will explore a finding that the clinically approved PS, methylene blue and related penothiazinium salts could have their antimicrobial PDT effect potentiated by addition of simple ions like iodide. While we initially interpreted this to involve electron transfer (oxidation) from hydroxyl radicals, we have now discovered that we can still get killing in the absence of oxygen. In addition to studying the contribution of Type 1 and Type 2 photochemical mechanisms we are now investigating a possible mechanism involving direct oxidation of iodide (and azide) anions by excited state MB to produce iodide/azide radicals that efficiently kill microbial cells. Aim 2 proposes a solution t the biggest barrier to the effectiveness of antimicrobial PDT in vivo i. e. delivering the photosensitizer (PS) into the infected tissue. Since all highly effective antimicrobial PS have cationic charges they are ideally suited for delivery into tissue by electricity. We will explore te use of iontophoresis and electroporation singly and in combination to deliver antimicrobial PS (and iodide) into ex vivo pigskin and into hairless mouse skin. Aim 3 responds to the reviewers' criticisms by exploring the combination of PDT with traditional systemic antibiotics to prevent regrowth of bacteria after PDT. Preliminary data has shown that a sub-therapeutic regimen of tobramycin can synergistically combine with a PDT regimen mediated by a fullerene and white light to save mice from dying in a Pseudomonas wound infection model. We will study possible synergy in vitro using MIC determinations with PDT and/or antibiotics In aim 4 in response to the reviewers' criticisms we will study selectivity for killing microbial cells (both fungi and bacteria) over mammalian skin cells (both mouse and human) and look at possible damage in tissue removed from mice. We have found some highly effective antifungal PS and will study these in vitro and in vivo with emphasis on selectivity. We now have access to Candida albicans that has been genetically engineered to express Gaussia princeps luciferase and Aspergillus fumigates expressing firefly luciferase that form localized infections that can be imaged by bioluminescence imaging.